Deep sequencing identifies new and regulated microRNAs in Schmidtea mediterranea

Abstract

MicroRNAs (miRNAs) play important roles in directing the differentiation of cells down a variety of cell lineage pathways. The planarian Schmidtea mediterranea can regenerate all lost body tissue after amputation due to a population of pluripotent somatic stem cells called neoblasts, and is therefore an excellent model organism to study the roles of miRNAs in stem cell function. Here, we use a combination of deep sequencing and bioinformatics to discover 66 new miRNAs in S. mediterranea. We also identify 21 miRNAs that are specifically expressed in either sexual or asexual animals. Finally, we identified five miRNAs whose expression is sensitive to gamma-irradiation, suggesting they are expressed in neoblasts or early neoblast progeny. Together, these results increase the known repertoire of S. mediterranea miRNAs and identify numerous regulated miRNAs that may play important roles in regeneration, homeostasis, neoblast function, and reproduction.

FIGURE 1.

Sample quality control. (A) FACS analysis to monitor the neoblast population in untreated or γ-irradiated animals. The fold change of the X1, X2, and Xins populations in the irradiated sample compared with the untreated sample is graphed. The fold change of the Xins population was arbitrarily set to a value of 1.0 and the values for X1 and X2 adjusted accordingly. (B) Analysis of smedwi-1 mRNA level. The level of smedwi-1 mRNA, a neoblast marker (Reddien et al. 2005), was analyzed using semiquantitative RT-PCR. As a control, a parallel experiment was performed for smed-actin mRNA.

FIGURE 2.

Deep sequencing of planarian miRNAs. (A) Alignment of sequence reads to pre-miR-755. The most frequently obtained mature, loop, and miRNA* strand sequences are highlighted in red, blue, and green, respectively, and the number of total reads obtained for each sequence are shown on the right. The location of these sequences on the secondary structure of the pre-miRNA is depicted below the alignment. (B) Histograms of the MIResque scores for known human miRNAs, all S. mediterranea loci containing clusters of deep sequence reads, known S. mediterranea miRNAs, and new S. mediterranea miRNAs identified in this study. The dotted line indicates the threshold at which a genomic region is considered to be a candidate miRNA locus. (D) Northern blot validation of MIResque predicted miRNAs. The structure for each pre-miRNA is shown on the right.

FIGURE 3.

Strain-specific miRNA Expression. (A) MicroRNAs that are expressed at least twofold higher in sexual animals than in asexual animals were identified, and the expression ratios were log₂ transformed and depicted in heat map representation. (B) MicroRNAs that are expressed at least twofold higher in asexual animals than in sexual animals were identified, and the expression ratios were log₂ transformed and are depicted in heat map representation. (C) Northern blots for mir-754b-1 and mir-2160-1 were performed with total RNA isolated from intact (−) and irradiated (+) sexual and asexual animals. The microRNA mir-71c was used as a probe to control for equal RNA loading.

FIGURE 4.

Irradiation-sensitive miRNA Expression. (A) MicroRNAs whose levels are reduced at least twofold after irradiation were identified and the expression ratios were log₂ transformed and depicted in heat map representation. (B) Northern blots for mir-13 and let-7a were performed with total RNA isolated from intact (−) and irradiated (+) sexual and asexual animals. The microRNA bantam-a was used as a probe to control for equal RNA loading. (C) Schematic depiction of the organization of the mir-71b/mir-2d/mir-752/mir-13 miRNA cluster (red, mature strand; blue * strand).